AU2020224470A1 - Hemagglutinin-binding peptide - Google Patents

Abstract

[Problem to be solved] To provide a compound having markedly higher antiviral activity than iHA 100, an intermediate for producing the compound, and a medical drug, or the like, containing the high-activity compound above. [Solution] The invention relates to a hemagglutinin-binding peptide, a pharmaceutically acceptable salts, or a solvate thereof, the hemagglutinin-binding peptide being: (1) a polypeptide comprising the amino acid sequence represented by sequence no. 1 or 2: Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys (sequence no. 1), Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys (sequence no. 2), or the like.

Description

DESCRIPTION HEMAGGLUTININ-BINDING PEPTIDE TECHNICAL FIELD

[0001] The present invention relates to a hemagglutinin

binding peptide having extremely high anti-influenza virus

activity, a medical drug for the prevention or therapy of

influenza, and a detection agent for influenza detection,

etc.

BACKGROUND ART

[0002] Influenza viruses are highly infectious and

pathogenic viruses, and their epidemics show widespread

pathogenicity to humans as a pandemic.

[0003] Zanamivir (brand name: Relenza(trademark)),

oseltamivir (brand name: Tamiflu(trademark)), peramivir

(brand name: Rapiacta(trademark)), and laninamivir (brand

name: Inavir(trademark)), which inhibit neuraminidase that

is necessary for the release of an influenza virus, are

widely used as pharmaceutical products against influenza

viruses. In addition, amantadine (brand name:

Symmetrel(trademark)) and flumadine (brand name:

Rimantadine(trademark)), which inhibit the enucleation

process of viruses, and baloxavir marboxyl (brand name:

Zofluza(trademark)), which inhibits cap-dependent

endonuclease, are known as pharmaceutical products. However, although the above pharmaceutical products, especially anti influenza drugs against neuraminidase, are widely used as pharmaceutical products, drug resistance due to mutation of viruses becomes a problem.

[0004] On the other hand, hemagglutinins are known as an

essential protein for an entry of an influenza virus into a

host cell, and an anti-virus molecule against this target

has been reported. An anti-virus molecule that targets

hemagglutinins is effective against a virus that is drug

resistant as an existing anti-virus drug. Therefore, an

anti-virus molecule against a target molecule different from

existing pharmaceutical products is also highly useful from

the viewpoint of drug resistance.

[0005] As an anti-virus molecule against hemagglutinins, an

antibody molecule that binds to a hemagglutinin has been

reported (e.g., Patent Documents 1-3). On the other hand,

while antibody pharmaceutical products have high activity,

a neutralizing antibody, which are generated when an antibody

pharmaceutical product is recognized as a foreign substance

in vivo, may significantly reduce the titer of the drug.

[0006] Recently, a peptide molecule having a special skeleton

such as an N-methyl amino acid and a D-amino acid has been

reported as a new group of molecules, which not only show

high binding power and biological stability, but also their molecular weight is extremely small compared to antibodies.

Therefore, it is attracting attention as a group of molecules

that may solve problems faced by conventional antibody

pharmaceutical products (e.g., Non-Patent Documents 1-4).

[0007] We have focused on the peptide molecule having the

special skeleton, and have been searching for a peptide

molecule against hemagglutinins. iHA100, a peptide molecule

against hemagglutinins, is an anti-influenza virus molecule

that exhibits anti-virus activity even when administered

intranasally, which differs greatly from its molecular

weight and the usual route of antibody administration (Patent

Document 4). On the other hand, there are no reports of

cases of significant improvement in anti-influenza virus

activity of iHA100 and the structure thereof, and the

improvement of anti-influenza virus activity thereof may

greatly contribute to an improvement of a drug effect.

PRIOR ART DOCUMENTS PATENT DOCUMENTS

[0008] Patent Document 1: International Publication No.

2018/108086 Brochure

Patent Document 2: International Publication No.

2018/015012 Brochure

Patent Document 3: International Publication No.

2017/122087 Brochure

Patent Document 4: International Publication No.

2013/071904 Brochure

Non-Patent Document 1: Nature Reviews Drug Discovery

17, 531-533 (2018)

Non-Patent Document 2: Current opinion in chemical

biology, 34, 44-52 (2016)

Non-Patent Document 3: Annual Review of Biochemistry,

83, 727-752 (2014)

Non-Patent Document 4: Chemistry, 19, 6530-6536 (2013)

SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] The purpose of this invention is to provide a compound

having significantly higher anti-virus activity than iHA100,

an intermediate for producing the compound, and a medical

drug containing the above high-activity compound, etc.

MEANS TO SOLVE THE PROBLEMS

[0010] The present invention is based on the finding that a

newly synthesized hemagglutinin-binding peptide has

remarkable activity compared to a previously known

hemagglutinin-binding peptide having anti-influenza virus

activity.

[0011] One of the embodiments disclosed herein relates to a

hemagglutinin-binding peptide, a pharmaceutically acceptable

salt thereof, or a solvate thereof (these are also referred

to as peptides of the present invention).

[0012] This hemagglutinin-binding peptide is any of the

following peptides (1) to (7):

(1) a polypeptide consisting of an amino acid sequence

represented by the sequence no. 1 or 2:

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr

Val-hydPro-Ala-Cys (sequence no. 1), and

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr

Val-hydPro-Ala-Cys-Lys (sequence no. 2);

(2) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1 or 2, the N-terminal Trp is

chloroacetyl-Trp;

(3) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the N-terminal Trp is

chloroacetyl-Trp and the C-terminal Cys has been modified as

represented by Formula (I) via an amide bond;

(4) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the N-terminal Trp is chloroacetyl

Trp and the C-terminal Lys has been substituted with a

modified lysine derivative represented by Formula (II);

(5) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the C-terminal Cys has been

modified as represented by Formula (I) via an amide bond;

(6) a polypeptide consisting of a sequence including

a lysine derivative represented by Formula (II) in which, in

the sequence no. 2, the side chain of Lys has been modified

with an acyl group; and

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1) to (6) above, one

or two amino acids have been deleted, added, substituted, or

inserted (however a sequence in which, in the sequence no.

1, the C-terminal Cys has been deleted, and a sequence in

which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded)

[0013] Formula (I)

[Formula 9]

H 1 A N H 0

(In Formula (I), * denotes a linking moiety to a carbonyl

group of the C-terminal Cys, and A' denotes a C8-C12 alkyl

group.)

[0014] Formula (II)

[Formula 10]

0 NH 2

N NH H A O

(In Formula (II), * denotes a linking moiety to a carbonyl

group of the C-terminal Cys, and A' denotes a C8-C12 alkyl

group.)

[0015] Preferred examples of the hemagglutinin-binding

peptide are as follows:

(1) the polypeptide consisting of the amino acid

sequence represented by the sequence no. 1 or 2;

(5) the polypeptide consisting of the amino acid

sequence in which, in the sequence no. 1, the C-terminal Cys

has been modified as represented by Formula (I) via an amide

bond;

(6) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the C-terminal Lys has been

substituted with a modified lysine derivative represented by

Formula (II); or

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1), (5) and (6) above,

one or two amino acids have been deleted, added, substituted,

or inserted (however the sequence in which, in the sequence

no. 1, the C-terminal Cys has been deleted, and the sequence

in which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded).

[0016] A preferred example of the hemagglutinin-binding

peptide is one in which the hemagglutinin-binding peptide is

cyclic.

[0017] A preferred example of the hemagglutinin-binding

peptide is one represented by formula (III) or (IV) below.

[0018] [Formula 11]

OH OH

N H NH N N HNN H H 0 0 0 OH 0 NH

0 NH 0 0 -- 1Y

N NH HN A2 A C NH

HN 0 0 N O OH HH 0

N o HN 0

NN 0 N H HY 0 0 OH

(In Formula (III), Formula A 2 denotes a group represented by

-NH 2 or a group represented by Formula (I).)

[0019] [Formula 12]

0 OH 3 A S -- y 0 HNH

H O NHN O NHN

/N: HO 0 NH HO O (IV)

NH OH N 0

00

0 NH HO HN HNH

N H N 0 r H 0 N

N

(In Formula (IV), Formula A 3 denotes a group represented by

-NH 2 or a group represented by Formula (II).)

[0020] A preferred example of the hemagglutinin-binding

peptide other than the above is represented by formula (V)

or (VI) below.

[0021] [Formula 13]

OH HH

NH N N HN N H H 0 0 0 "'"OH 0 NH

0 NH 0

N 0¼ /NH A0(

OH HN HH NA N O H O OH

(In Formula (V) ,Formula A4 denotes a group represented by

NH2 or a group represented by Formula (IIa) .)

[0022] [Formula 14]

O NH2

N NH H

AlO

* (In Formula (IIa), denotes a linking moiety, and Al denotes

a C-C12 alkyl group.)

[0023] [Formula 15]

0 OH

O5 N A HN H H O NH N HN

HN HO 0 NH

0 Y0 HOIIl'.'" N N (VI)

NH OH N O

0 0 HO HN NH __N 0

N N N 0 H o N

N

In Formula (VI), Formula A 5 denotes a group represented by

NH 2 or a group represented by Formula (I).

[0024] A preferred example of the hemagglutinin-binding

peptide other than the above is represented by formula (VII)

below.

[Formula 16]

0

HN O OH

O NHS 0 N HN 0 HN ''HO NH

HO' o N 0 N

NH OH N 0 Hr 0 0J

H HO 0 N -'.NH N 0 NT HH

N 0

N

[0025] An aspect in this description other than the above is

a medical drug for the prevention of virus infection or for

the therapy of a virus infectious disease including any of

the above-mentioned hemagglutinin-binding peptide,

pharmaceutically acceptable salt thereof, or solvate thereof.

Yet another aspect is a medical drug for the prevention or

therapy of influenza (a prophylactic agent or therapeutic

agent, a pharmaceutical product for influenza) including any

of the above-mentioned hemagglutinin-binding peptide,

pharmaceutically acceptable salt thereof, or solvate thereof.

[0026] Another embodiment disclosed in this description relates to a virus detection agent. Yet another aspect relates to an influenza virus detection agent. This virus detection agent contains the above-mentioned hemagglutinin binding peptide.

[0027] Another embodiment disclosed in this description

relates to a kit for virus detection contains the above

mentioned virus detection agent.

EFFECTS OF THE INVENTION

[0028] As shown by the examples, this application may provide

a peptide having significantly higher anti-influenza virus

activity than iHA100, a medical drug for the prevention or

therapy of influenza and an influenza virus detection agent

using the peptide, and an intermediate for synthesizing the

above-mentioned peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1-1 are graphs showing evaluation results of

in vitro anti-influenza virus activity of iHA100 and HA152

using influenza virus A/Nagasaki/HA-58/2009 (H1N1).

Figure 1-2 are graphs showing evaluation results of in

vitro anti-influenza virus activity of iHA100 and HA152 using

influenza virus A/Puerto Rico/8/34 (H1N1).

Figure 1-3 are graphs showing evaluation results of in

vitro anti-influenza virus activity of iHA100 and HA152 using

influenza virus A/Duck/Pennsylvania/84 (H5N2).

Figure 2 is a table showing results of in vitro anti

influenza virus activity for iHA100, and HA119, HA145, HA146,

HA151, HA152 using influenza virus A/Duck/Pennsylvania/84

(H5N2) as EC50 value.

Figure 3 is a graph showing analysis result of in vivo

anti-influenza virus activity of HA152 using the influenza

virus A/Puerto Rico/8/34 (H1N1) infection model.

EMBODIMENTS TO CARRY OUT THE INVENTION

[0030] Embodiments to carry out the present invention will

be described below. The present invention is not limited to

the embodiments described below, and includes those

appropriately modified by those skilled in the art from the

following embodiments to the extent obvious.

[0031] One of the embodiments disclosed herein relates to a

hemagglutinin-binding peptide, a hemagglutinin-binding

peptide, a pharmaceutically acceptable salt thereof, or a

solvate thereof.

[0032] Herein, the hemagglutinin means an antigenic

glycoprotein present on the surface of many bacteria and

viruses, including influenza viruses, and is denoted as "HA".

The hemagglutinin is involved in a process of virus adhesion

to a host cell. Specifically, when the hemagglutinin on the

surface of the virus binds to a sialic acid on the surface

of the target host cell, the virus is enveloped by a cell membrane and taken up into the cell in the form of a virus containing endosome. Subsequently, an endosomal membrane and a virus membrane fuse, a virus genome is inserted into the cell, and proliferation begins.

Influenza viruses are classified into three types,

type A, type B, and type C. There are at least 16 subtypes

of hemagglutinins in influenza virus A type that are

particularly prone to pandemics, and they are referred to as

Hi to H16. Hi, H2, H5, H6, H8, H9, Hl, H12, H13, H16, H17,

H18 are referred to as Group I, and the other hemagglutinins

(H3, H4, H7, H10, H14, H15) are referred to as Group II.

The H in the subtype name of influenza indicates the

hemagglutinin.

[0033] The hemagglutinin-binding peptide means a peptide

that may bind to the hemagglutinin. Whether or not it may

bind to the hemagglutinin may be confirmed according to a

method known to those skilled in the art.

[0034] The pharmaceutically acceptable salt thereof means a

pharmaceutically acceptable salt of the hemagglutinin

binding peptide. Examples of the salt include addition salts

of inorganic acids (hydrochloric acid, hydrobromic acid,

hydriodic acid, sulfuric acid, phosphoric acid, etc.),

addition salts of organic acids (p-toluene sulfonic acid,

methane sulfonic acid, oxalic acid, p-bromophenyl sulfonic

acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide, or alkaline or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), addition salts of amino acids.

[0035] The pharmaceutically acceptable solvate thereof means

a pharmaceutically acceptable solvate of the hemagglutinin

binding peptide, or a pharmaceutically acceptable solvate of

the hemagglutinin-binding peptide salt. A Solvent molecule

may be coordinated to the compound or the salt thereof, and

examples of the solvate are hydrates and alcoholates.

[0036] This hemagglutinin-binding peptide is any of the

following peptides (1) to (7):

(1) a polypeptide consisting of an amino acid sequence

represented by the sequence no. 1 or 2:

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr

Val-hydPro-Ala-Cys (sequence no. 1), and

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr

Val-hydPro-Ala-Cys-Lys (sequence no. 2);

(2) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1 or 2, the N-terminal Trp is

chloroacetyl-Trp;

(3) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the N-terminal Trp is

chloroacetyl-Trp and the C-terminal Cys has been modified as

represented by Formula (I) via an amide bond;

(4) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the N-terminal Trp is chloroacetyl

Trp and the C-terminal Lys has been substituted with a

modified lysine derivative represented by Formula (II);

(5) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the C-terminal Cys has been

modified as represented by Formula (I) via an amide bond;

(6) a polypeptide consisting of a sequence including

a lysine derivative represented by Formula (II) in which, in

the sequence no. 2, the side chain of Lys has been modified

with an acyl group; and

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1) to (6) above, one

or two amino acids have been deleted, added, substituted, or

inserted (however a sequence in which, in the sequence no.

1, the C-terminal Cys has been deleted, and a sequence in

which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded)

[0037] Formula (I)

[Formula 17]

H N A1 N H 0

(In Formula (I), * denotes a linking moiety to a carbonyl group of the C-terminal Cys, and A' denotes a C8-C12 alkyl group.) The Cs- C12 alkyl group is an alkyl group with 8-12 carbons. The C8- C12 alkyl group may be a linear alkyl group or a branched alkyl group. The Cs- C12 alkyl group may be any of Cs alkyl group, C9 alkyl group, Cio alkyl group, Cii alkyl group, and C12 alkyl group. The same applies to A' in each of the following groups.

[0038] Formula (II)

[Formula 18]

0 NH 2

N NH

HA 1 O

(In Formula (II), * denotes a linking moiety to a carbonyl

group of the C-terminal Cys, and A' denotes a C8-C12 alkyl

group.)

[0039] Preferred examples of the hemagglutinin-binding

peptide are as follows:

(1) the polypeptide consisting of the amino acid

sequence represented by the sequence no. 1 or 2;

(5) the polypeptide consisting of the amino acid

sequence in which, in the sequence no. 1, the C-terminal Cys has been modified as represented by Formula (I) via an amide bond;

(6) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the C-terminal Lys has been

substituted with a modified lysine derivative represented by

Formula (II); or

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1), (5) and (6) above,

one or two amino acids have been deleted, added, substituted,

or inserted (however the sequence in which, in the sequence

no. 1, the C-terminal Cys has been deleted, and the sequence

in which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded).

[0040] Examples of a specific amino acid sequence of this

peptide is as follows.

Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla

His-Tyr-Thr-Val-hydPro-Ala-Cys-NH2 (sequence no.: 3)

Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla

His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=0)n-C 1 1 H23]-NH 2

(sequence no.: 4)

Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla

His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=0)n-CHig]-NH2

(sequence no.: 5)

Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla

His-Tyr-Thr-Val-hydPro-Ala-Cys- [NHCH2 C H2NHC (=0) n-C1 1 H23]

(sequence no.: 6)

Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla

His-Tyr-Thr-Val-hydPro-Ala-Cys- [NHCH 2CH 2NHC (=0) n-CgHig]

(sequence no.: 7)

[0041] Herein, the amino acid (proteinogenic amino acid)

residues that comprise the peptides and the polypeptides of

the present invention, as well as comprise proteins, are

denoted using three-letter or single-letter notations

accepted by the industry.

[0042] Another amino acid disclosed herein includes amino

acids that do not comprise proteins (also referred to as a

non-proteinogenic amino acid, or simply a non-natural amino

acid), or chemically synthesized compounds having properties

known in the industry that are characteristic of amino acids.

Examples of the non-natural amino acid include, but are not

limited to, a,a-disubstituted amino acids (e.g., a-methyl

alanine), N-alkyl-a-amino acids, N-alkyl-a-D-amino acids, B amino acids, whose main chain structure differs from the

natural type, and amino acids whose side chain structure

differs from the natural type (e.g., norleucine,

homohistidine, and hydroxyproline).

[0043] As examples of the non-natural amino acid disclosed

herein, N-methyl glycine, a type of N-methyl amino acid that

is an N-alkyl-a-amino acid, may be denoted as MeGly, N-methyl

alanine as MeAla, and N-methyl phenylalanine as MePhe. In addition, as an example of the amino acid whose side chain structure differs from the natural type, 4R-hydroxyproline may be denoted as hydPro.

[0044] Chloroacetyl- means chloroacetylation, and

Chloroacetyl-Trp denotes chloroacetyl-Trp.

[0045] Herein, the polypeptide means one in which two or

more amino acids are bonded by a peptide bond, for example,

may be one in which 8-30 amino acids are bonded by peptide

bonds, and may be linear or cyclic. The polypeptide herein

is preferably a cyclic amino acid of 15 or 16 amino acids.

[0046] In addition, the hemagglutinin-binding peptides

according to the present invention may be cyclized

(macrocyclized). Herein, the cyclization means that within

a peptide, two amino acids separated by one or more amino

acids are bonded directly or indirectly via a linker, etc.

to make a cyclic structure within the molecule.

[0047] The cyclization may be carried out by a known method,

for example, according to the method described in

International Publication W02016/063969 brochure.

[0048] A preferred example of the hemagglutinin-binding

peptide is one represented by formula (III) or (IV) below.

[0049] [Formula 19]

OH OH

NH O N NH H 0 H 0 N 0 OH 0 NH 0 NH HNN N 0 A

/: NH A2 I)

S 0 HN 0 0 OHN0

()N0 O O HN OH0

0 NINH N HY 0 0 OH

(In Formula (III), Formula A 2 denotes a group represented by

-NH 2 or a group represented by Formula (I).)

[0050] [Formula 20]

3 0 OH A S 0 H HN 0

N

I-NX / HO 0 NH

HO I 0 1Y

rN 0 (N (IV) HO NN

>N 0 Y NH OH

HO NH

HOHN 0_NH

N N N 0 HJ HH o

y N

[0052]-- (In Formula (IV), Formula A 3 denotes a group represented by

[Frul01 -NH 2 or a group represented by Formula (TI).)

[0051] A preferred example of the hemagglutinin-binding

peptide other than the above is represented by formula (V)

or (VT) below.

[0052] [Formula 21]

OH OH

N H NH N N HN N H H 0 0 0 'OH 0 NH 0 NH - 0

N 'N NHHNA HNN Nr" a 4 NH A (V)

O N

O OH O N01 HN : 0 N N 0 N C-H 0 0 OH

(In Formula (V), Formula A 4 denotes a group represented by

NH 2 or a group represented by Formula (IIa).)

[0053] [Formula 22]

0 NH 2

N NH

HA 1 0 i >

(In Formula (IIa), * denotes a linking moiety, and A' denotes

a C8-C 1 2 alkyl group.)

[0054] [Formula 23]

0 OH o S 0

HN 0 5 A N H O NH N

/ HO N " 0 HOII..- N N O ~N (VI)

NH OH N O

0 0 N NH HO HO

N O N

In Formula (VI), Formula A 5 denotes a group represented by

NH 2 or a group represented by Formula (I).

[0055] A preferred example of the hemagglutinin-binding

peptide other than the above is represented by formula (VII)

below.

[Formula 24]

0

HN

NH S 0 O H

HN H O NHN H N >HN 0 HN HO NH

HO" N, 0 N4

NHOH N 0

N N 0 N N 0

N

[0056] Specific structures of the cyclic peptide are as

follows. Hr

Ss H

[0057] [Formula O H- HN- 25] O

HO\

HO- N 0 N

NN O N 0 ',N 0. H0 HO N'zl" 0s H & 0; N

0Y 0O HOV IA 9

[0058] [Formula 26]

N" oHOO 0 NH H H

0 N 0H 0 HO N N -, >N N N N

H 2N 0 0 HN sHN O O IHO

Hd

[0059] [Formula 27] N "o N N

H HI

NN NN O OO NH 0 N N HO O N

H HH N N - HN ". N

° 2N H 0 0 HN0

H

(11A146)

[0060] [Formula 28]

O N H

Hs H N 0 0N H O

HN 0 HO, 0

0 HN0 NHHN"

HH HO

N~ OINl

[0061] [Formula 29] 0 N H 0 ,N N'

HN O HO" O

OH O O H O NN HN N H6

0 (HA15) H HN 0 HO H 0 NH 0 H 0 HON 0 HH 0r N HO NH'

[0062] Teptdsfh per N NH 0~~ HN,-, N HN0HO 0

HO

(II15l2)

[0062] The peptides of the present invention may be produced

by known methods of peptide production such as chemical

synthesis methods including liquid-phase methods, solid

phase methods, and hybrid methods combining the liquid-phase and the solid-phase methods; and genetic recombination methods.

[0063] The solid phase method is, for example, an

esterification reaction between a hydroxyl group of a resin

having the hydroxyl group and a carboxy group of the first

amino acid (usually, the C-terminal amino acid of the target

peptide) whose a-amino group is protected by a protecting

group. As esterification catalysts, known dehydration and

condensation agents such as 1-mesitylene sulfonyl-3-nitro

1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and

diisopropylcarbodiimide (DIPCDI) may be used.

Next, the protecting group of the a-amino group of the

first amino acid is removed, and the second amino acid, in

which all functional groups except the carboxy group of the

main chain are protected, is added to activate said carboxy

group, and the first and second amino acids are bonded.

Furthermore, the a-amino group of the second amino acid is

deprotected, and the third amino acid, in which all

functional groups except the carboxy group of the main chain

are protected, is added to activate said carboxy group, and

the second and third amino acids are bonded. This process

is repeated until a peptide of the desired length is

synthesized, and then all functional groups are deprotected.

[0064] The resin used in the solid phase method includes

Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin,

Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin

(Merck), HMPA-PEGA resin (Merck). These resins may be washed

with solvents (dimethylformamide (DMF), 2-propanol,

methylene chloride, etc.) before use.

The protecting groups for the a-amino group include,

but are not limited to, for example, benzyloxy carbonyl (Cbz

or Z) group, tert-butoxy carbonyl (Boc) group, fluorenyl

methoxycarbonyl (Fmoc) group, benzyl group, allyl group, and

allyloxy carbonyl (Alloc) group, as long as they are known

protecting groups. The Cbz group may be deprotected by

hydrofluoric acid, hydrogenation, etc., the Boc group may be

deprotected by trifluoroacetic acid (TFA), and the Fmoc group

may be deprotected by a treatment with piperidine.

The protecting groups for the a-carboxy group include,

but are not limited to, for example, methyl ester, ethyl

ester, benzyl ester, tert-butyl ester, cyclohexyl ester, as

long as they are known protecting groups.

As other functional groups of amino acids, for example,

the hydroxy group of serine and threonine may be protected

by benzyl group or tert-butyl group, and the hydroxy group

of tyrosine is protected by 2-bromobenzyloxycarbonyl group

or tert-butyl group, but not particularly limited. The amino

group of the side chain of lysine, and the carboxy group of

glutamic acid and aspartic acid may be protected in the same

way as the a-amino group and a-carboxy group.

[0065] Activation of the carboxy group may be carried out using a condensing agent. The condensing agents include, for example, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC or WSC), (1H benzotriazol-1-yloxy)tris(dimethyl amino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethyl amino)methyl]

1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0066] A cleavage of the peptide chain from the resin may be

carried out by a treatment with an acid such as TFA, hydrogen

fluoride (HF).

[0067] The production of peptides by the genetic

recombination method (a translation synthesis system) may be

carried out using a nucleic acid encoding the peptides of

the present invention. The nucleic acid encoding the

peptides of the present invention may be DNA or RNA.

The nucleic acid encoding the peptides of the present

invention may be prepared by a known method or a method

similar thereto. For example, it may be synthesized by an

automatic synthesis apparatus. A restriction enzyme

recognition site may be added to insert the resulting DNA

into a vector, or a base sequence encoding an amino acid

sequence may be incorporated to cut out the resulting peptide

chains by an enzyme, etc.

As mentioned above, when the peptide of the present

invention is fused with a membrane permeable peptide, etc., the above nucleic acid includes a nucleic acid encoding the membrane permeable peptide.

In order to inhibit degradation by a host-derived

protease, a chimeric protein expression method may be used

in which a target peptide is expressed as a chimeric peptide

with another peptide. In this case, a nucleic acid that

encodes the target peptide and a peptide that binds to this,

is used as the above nucleic acid.

[0068] Subsequently, an expression vector is prepared using

the nucleic acid encoding the peptides of the present

invention. The nucleic acid may be inserted into the

downstream of a promoter of the expression vector, either as

is, or by digesting it with a restriction enzyme, or adding

a linker, etc. The vectors include E. coli-derived plasmids

(pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118,

pBluescript II, etc.), Bacillus subtilis-derived plasmids

(pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast

derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.),

bacteriophages (ePhage, M13 Phage, etc.), viruses

(retrovirus, vaccinia virus, adenovirus, adeno-associated

virus (AAV), cauliflower mosaic virus, tobacco mosaic virus,

baculovirus, etc.), cosmid.

[0069] The promoter may be appropriately selected depending

on the type of host. If the host is an animal cell, for

example, SV40 (simian virus 40)-derived promoter or CMV

(cytomegalovirus)-derived promoter may be used. If the host

is E. coli, trp promoter, T7 promoter, lac promoter, etc.

may be used.

Nucleic acids encoding DNA replication start sites

(ori), selection markers (antibiotic resistance, nutritional

requirement, etc.) enhancers, splicing signals, poly(A)

addition signals, tags (FLAG, HA, GST, GFP, etc.), etc. may

also be incorporated into the expression vector.

[0070] Next, an appropriate host cell is transformed with

the above expression vector. The host may be appropriately

selected in relation to the vector, for example, E. coli,

Bacillus subtilis, Bacillus sp.), yeast, insect or insect

cells, animal cells, etc. are used. For example, HEK293T

cells, CHO cells, COS cells, myeloma cells, HeLa cells, and

Vero cells may be used as the animal cell. Transformation

may be carried out according to known methods such as

lipofection methods, calcium phosphate methods,

electroporation methods, microinjection methods, particle

gun methods, depending on the type of host. The target

peptide is expressed by culturing the transformant according

to the usual method.

[0071] Purification of the peptides from the transformant

culture includes collecting the cultured cells, suspending

them in an appropriate buffer solution, then destroying the

cells by methods such as sonication, freeze-thawing, and obtaining a crude extract by centrifugation or filtration.

If the peptide is secreted into the culture medium, the

supernatant is collected.

Purification from the crude extract or the culture

supernatant may also be carried out by known methods or

methods similar thereto (e.g., salting out, dialysis methods,

ultrafiltration methods, gel filtration methods, SDS-PAGE

methods, ion exchange chromatography, affinity

chromatography, and reverse phase high performance liquid

chromatography).

The resulting peptide may be converted from an educt

to a salt or from a salt to an educt by a known method or a

method similar thereto.

[0072] The translation synthesis system may be a cell-free

translation system. The cell-free translation system

includes, for example, ribosomal proteins, aminoacyl-tRNA

synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP,

translation initiation factors (IF), elongation factors (EF),

release factors (RF), and ribosomal regeneration factors

(RRF), as well as other factors required for translation.

E. coli extract and wheat germ extract may be added to

increase the efficiency of expression. In addition, rabbit

red blood cell extract or insect cell extract may be added.

Several hundred pg to several mg/mL of the peptide may

be produced by continuously supplying energy to the system

containing these peptides using dialysis. The system may include RNA polymerase to carry out transcription from genetic DNA as well. As commercially available cell-free translation systems, RTS-100 (registered trademark) from

Roche Diagnostics, PURESYSTEM from Gene Frontier, and

PURExpress In Vitro Protein Synthesis Kit from NEW ENGLAND

Biolabs, etc. may be used as a system derived from E. coli,

and those of Zoigene and CellFree Sciences may be used as a

system using wheat germ extract.

According to the cell-free translation system, the

expression product may be obtained in a highly pure form

without purification.

[0073] In the cell-free translation system, instead of

aminoacyl-tRNA synthesized by a natural aminoacyl-tRNA

synthetase, an artificial aminoacyl-tRNA, in which a desired

amino acid or hydroxy acid is linked (acylated) to the tRNA,

may be used. Such aminoacyl-tRNA may be synthesized using

an artificial ribozyme.

Such ribozymes include flexizyme (H. Murakami, H.

Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10,

655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003),

Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A.

Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357

359 "The flexizyme system: a highly flexible tRNA

aminoacylation tool for the synthesis of nonnatural

peptides"; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009)

Bioorganic & Medicinal Chemistry Letters 19, 3892-3894 "A flexizyme that selectively charges amino acids activated by a water-friendly leaving group"; and W02007/066627, etc.)

. Flexizyme is also known by names such as the original

flexizyme (Fx), and modified versions of it, such as dinitro

benzyl flexizyme (dFx), enhanced flexizyme (eFx), and amino

flexizyme (aFx).

[0074] By using tRNA linked with the desired amino acid or

hydroxy acid produced by the flexizyme, a desired codon may

be translated in association with the desired amino acid or

hydroxy acid. A Special amino acid may be used as the

desired amino acid. For example, the above-mentioned non

natural amino acids required for the cyclization may also be

introduced into the hemagglutinin-binding peptide by this

method.

[0075] Chemical synthesis of the macrocyclic peptides and

analogs thereof of the present invention may be synthesized

using various methods commonly used in the art, including

stepwise solid-phase synthesis, semi-synthesis of a peptide

fragment via conformationally supported religation, and

chemical ligation. The synthesis of the peptides and the

analogs thereof described herein is a chemical synthesis

using various solid phase techniques as described in, for

example, K. J. Jensen, P. T. Shelton, S. L. Pedersen, Peptide

Synthesis and Applications, 2nd Edition, Springer, 2013, etc.

The preferred strategy is based on the combination of an

Fmoc group that temporarily protects the a-amino group and

allows selective removal by a base, and a protecting group

that temporarily protects a side chain functional group and

is stable under deFmoc conditions. Such a general selection

of peptide side chains is known for the above-mentioned

Peptide Synthesis and Applications, 2nd edition, and G. B.

Fields, R. L. Noble, Solid Phase Peptide Synthesis Utilizing

9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide

Protein Res. 35, 1990, 161-214, etc., however, preferred

peptide side chain protecting groups include the Boc group

and Mtt group for lysine and other amino groups, the tert

butyl group for the carboxyl group of glutamic acid and

aspartic acid, and Trt group and Mmt group for the thiol

group of cysteine.

[0076] The synthesis of the resin as a precursor for peptide

synthesis in the present invention (or simply referred to as

"resin") may be prepared by using, for example, commercially

available PAL-PEG-resin and PAL-PEG-PS, etc., with reference

to the reported examples such as Biopolymers 2011;96(6):715

22. For example, an outline is shown in Scheme 1, wherein

n denotes the length of the carbon number, R1 denotes

hydrogen or an alkyl group with 1-4 carbons, R2 denotes

various alkyl chains or alkyl chains having a substituent.

The amino group of the resin 1 is sulfonylated with o-Ns-Cl

in the presence of an appropriate base to prepare 2. The

solid phase resin 4 may be obtained by reacting an amino primary alcohol 3 protected at the N-terminus with Fmoc such as Fmoc-glycinol, with a reagent used in combination with a

Mitsunobu-type reaction such as triphenylphosphine, in the

presence of diisopropyl azodicarboxylate, etc. Fmoc may be

deprotected by a combination of an appropriate secondary

amine in a solvent, such as DMF solution of piperidine, to

yield 5, and various acyl groups may be introduced into the

regenerated amine using a known peptide coupling method to

yield 6. As for R 2 , it may be introduced, for example, by

reacting decanoic acid having a long chain alkyl group

(composition formula CH3(CH2)8COOH) in the presence of the

condensing agents HATU and DIPEA. Finally, the resin 7, a

precursor for the peptide synthesis, may be adjusted by

treating the removal of the o-Ns group with a combination of

an appropriate base and a thiol, such as DBU and DODT.

[0077] [Formula 30]

Scheme 1 = ren M.

O-S-e TN-,bsas E Ns -- Fr A1 HR~r~akl R ~ 1 Foc N

Me CH2Cl MG 0DIAD, PPhM I 2 4 (PA-PEG EESE

1 R GZe RCOOH, R CMe R OMe piperidiniDMF H1 -4 ppidec uplngrengen(s N tho,basb R NI

5 6 7

[0078] In the peptide synthesis in the present invention, a

commercially available resin may be used as another method.

For example, an outline is shown in scheme-2. As another way

to adjust one preferred precursor resin, commercially available Rink Amide MBHA resin (Sigma-Aldrich) , Fmoc-Rink

Amide NovaPEG resin (Merck Millipore) , or Fmoc-NH-SAL-PEG

resin (Watanabe Chemical Industries, LTD.) was used as a

solid phase resin (8), after removal of Fmoc, Fmoc amino

acid 9 having a primary or secondary amino group on the side

chain having a protecting group that may be removed under

mild acidic conditions, such as Fmoc-Lys(Mtt)-OH (m=3, R 3=H,

S-configuration), may be condensed to obtain 10. The

resulting solid-phase resin may be treated with a solution

of TFA/TIS/CH 2 Cl 2 in a volume ratio of 1:4:95 to selectively

remove Mmt group to yield 11. Subsequently, after the

introduction of R 2 COOH as described above, 12 is yielded,

and with the removal of the Fmoc group, 13 may be yielded,

which may be used for the synthesis of the target peptide.

[0079] [Formula 31]

SV- 2 04 Fnc M R Hr-Cakyl F , •

F N 1) 20%pipid N1

10 11

F N ,N-4

12 13

[0080] The peptides and analogs thereof described in the

present invention may be synthesized in a stepwise method on

the solid phase resin as described above. In the C-terminal

amino acid used, and in all amino acids and peptides used in the synthesis, the a-amino protecting group must be selectively removed during the synthetic process.

Preferably, using the above-mentioned solid phase resin, the

C-terminal carboxyl group of a peptide whose N-terminus is

appropriately protected using a protecting group such as

Fmoc, or the C-terminal carboxyl group of an amino acid

appropriately protected using a protecting group such as

Fmoc, is made into an activated ester by an appropriate

reagent and then added to an amino group on the solid phase

resin to initiate the process. Subsequent elongation of the

peptide chain may be achieved by sequentially repeating the

removal of the N-terminal protecting group (e.g., Fmoc group),

followed by condensation of the protected amino acid

derivative, according to the amino acid sequence of the

target peptide. These may liberate the target peptide in

the final stage. For example, as a condition for liberation,

it may be liberated with a TFA solution containing

water/silyl hydride/thiol as a scavenger in TFA, which is

described in Teixeira, W.E. Benckhuijsen, P. E. de Koning,

A. R. P. M. Valentijn, J. W. Drijfhout, Protein Pept. Lett.,

2002, 9, 379-385, etc. A typical example is

TFA/Water/TIS/DODT (volume ratio 92.5:2.5:2.5:2.5).

[0081] The synthesis of the peptide analogs described herein

may be carried out by using a single or multi-channel peptide

synthesizer, such as CEM's Liberty Blue synthesizer or

Biotage's Syro I synthesizer.

[0082] Activation of the carboxy group may be carried out

using a condensing agent. The condensing agents include,

for example, dicyclohexylcarbodiimide (DCC),

diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3

dimethylaminopropyl) carbodiimide (EDC or WSC), (1H

benzotriazol-1-yloxy)tris(dimethyl amino)phosphonium

hexafluorophosphate (BOP), 1-[bis(dimethyl amino)methyl]

1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0083] Another embodiment disclosed in this description

relates to a medical drug. This medical drug includes the

above-mentioned hemagglutinin-binding peptides, the

pharmaceutically acceptable salts thereof, or solvates (for

brevity, these will be referred to as simply hemagglutinin

binding peptides below). The medical drug preferably

contains an effective amount of the above-mentioned

hemagglutinin-binding peptide as an active ingredient.

[0084] When the hemagglutinin-binding peptide is used for a

medical drug application, one or more of the amino acids in

the peptide may be chemically modified, for example, with a

hydrocarbon, a fatty acid, and polyethylene glycol (PEG),

etc., for functionalizing and various modifications. In

addition, these chemical modifications may also be carried

out using a linker. These modifications may, for example,

yield a peptide that are more chemically and metabolically stable. The linker is a substructure that allows a cyclic peptide that exhibits anti-virus activity (e.g., the basic structure shown in (II)) and chemical modification for functionalizing. Specific examples of the basic structure in the present invention are the 1,2-ethylenediamine structure represented in (I) and the lysin amide structure with NH 2 at the C-terminus represented in (II).

[0085] As shown in the examples below, the peptides of the

present invention exhibit anti-virus activity by binding to

a hemagglutinin on the surface of the virus. The type of

virus is not particularly limited, as long as it exhibits

anti-virus activity against viruses that infect humans, etc.

via a hemagglutinin or a similar protein. The type of virus

is, for example, an envelope virus, and more preferably, a

virus having a class I fusion protein. Specific examples of

the virus include seasonal influenza viruses (including

human, avian, and swine influenza viruses or novel influenza

viruses, also referred to as simply an influenza virus),

highly pathogenic avian influenza viruses, swine epidemic

diarrhea viruses, HIV-1, Ebola viruses, yellow fever viruses,

and corona viruses. The target virus is preferably the

influenza virus. In addition, the target influenza virus

may be any of the A, B, and C types, but preferably the A

type, and more preferably an influenza virus having the

hemagglutinin belonging to Group I. Herein, anti-influenza

virus activity is also referred to as anti-virus activity.

Compositions containing the peptides of the present

invention are useful as a preventive drug for virus infection

or a therapeutic drug for virus infectious diseases,

preferably as a preventive or therapeutic agent for influenza,

and similarly, the peptides of the present invention are

useful in a method of preventing virus infections or treating

virus infectious diseases, preferably in methods of

preventing or treating influenza.

[0086] Herein, the term "influenza" means an acute infectious

disease caused by the influenza virus. Infection with the

influenza virus causes cold-like symptoms with high fever,

muscle pain, etc., in humans. It may be accompanied by

gastrointestinal symptoms such as abdominal pain, vomiting,

and diarrhea, and complications include pneumonia and

influenza encephalopathy.

Herein, the term "infection" is used to refer to either

the process by which a virus invades a living body through

the skin or mucous membrane, or the process by which a virus

enters a cell through membrane fusion. In addition, herein,

the term "virus infection" means the state in which a virus

has invaded a living body, regardless of the presence or

absence of symptoms. In addition, herein, the term

"infectious disease" means various symptoms caused by a virus

infection.

[0087] Herein, the term "therapy or prevention of influenza" is used in its broadest sense, and means, for example, relieving or preventing the worsening of one or more symptoms associated with influenza virus infection, suppressing the development of symptoms after infection, preventing

(delaying or stopping) the infection of cells with the virus

in vivo, preventing (delaying or stopping) the proliferation

of the virus in vivo, a decrease in the number of viruses in

vivo, etc. If at least one of these is effective, it is

considered useful in the therapy or prevention of influenza.

In addition, as shown in the examples below, the

peptides of the present invention have neutralizing activity

against the hemagglutinins, so it is understood that the

same effect as influenza vaccine may be obtained.

[0088] Herein, the dosage form of the medical drug

composition is not particularly limited and may be oral

administration or parenteral administration. The parenteral

administration includes, for example, injection

administration such as intramuscular injection, intravenous

injection, and subcutaneous injection, transdermal

administration, and transmucosal administration (transnasal,

transoral, transocular, transpulmonary, transvaginal, and

transrectal) administration.

[0089] The above medical drug compositions may be modified

in various ways in consideration of the property of the

polypeptide to be easily metabolized and excreted. For example, polyethylene glycol (PEG) or a sugar chain may be added to the polypeptide to increase the residence time in the blood and reduce the antigenicity. In addition, bio degradable polymer compounds such as poly (lactic-co glycolic acid) (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc.

may be used as sustained release base agents, and the

polypeptide may be encapsulated in them. If administered

transdermally, a weak electric current may be applied to the

skin surface to penetrate the stratum corneum (iontophoresis

method).

[0090] The above medical drug compositions may be used as

the active ingredients as they are, or may be formulated by

adding pharmaceutically acceptable carriers, excipients,

additives, etc. Examples of dosage forms include liquids

(e.g., injections), dispersants, suspensions, tablets,

rounds, powders, suppositories, sprays, fine granules,

granules, capsules, syrups, lozenges, inhalants, ointments,

eye drops, nasal drops, ear drops, and papules.

The formulation may be carried out by a conventional

method, for example, using an excipient, a binder, a

disintegrant, a lubricant, a dissolving agent, a dissolution

aid, a colorant, a taste and odor correcting agent, a

stabilizer, an emulsifier, an absorption enhancer, a

surfactant, a pH adjuster, a preservative, an antioxidant, etc. as appropriate.

Examples of ingredients used in formulation include,

but are not limited to, pharmaceutically acceptable organic

solvents such as purified water, saline solution, phosphate

buffer solution, dextrose, glycerol and ethanol, animal and

vegetable oils, lactose, mannitol, glucose, sorbitol,

crystal cellulose, hydroxypropyl cellulose, starch,

cornstarch, silicic acid anhydride, magnesium aluminum

silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone,

carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium

polyacrylate, sodium alginate, water-soluble dextran, sodium

carboxymethyl starch, pectin, methyl cellulose, ethyl

cellulose, xanthan gum, gum arabic, tragacanth, casein, agar,

polyethylene glycol, diglycerin, glycerin, propylene glycol,

vaseline, paraffin, octyl dodecyl myristate, isopropyl

myristate, higher alcohol, stearyl alcohol, stearic acid,

human serum albumin.

In consideration of the fact that the peptide is

difficult to be absorbed through the mucosa, the above

medical drug compositions may contain an absorption enhancer

that improve absorption of poorly absorbable drugs. As such

absorption enhancers, surfactants such as polyoxyethylene

lauryl ethers, sodium lauryl sulfate, and saponins; bile

salts such as glycocholic acid, deoxycholic acid, and

taurocholic acid; chelating agents such as EDTA and salicylic

acids; fatty acids such as caproic acid, capric acid, lauric

acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acylamino acids, cyclodextrins, chitosan, nitric oxide donor, etc. may be used.

[0091] The rounds or tablets may also be coated with sugar

coating, gastric soluble and enteric soluble substances.

Injectable formulations may contain distilled water

for injection, physiological saline solution, propylene

glycol, polyethylene glycol, vegetable oils, alcohols, etc.

Furthermore, a wetting agent, an emulsifier, a dispersant,

a stabilizer, a dissolving agent, a dissolving aid, a

preservative may be added.

[0092] If the medical drug compositions of the present

invention is administered to mammals (e.g., humans, mice,

rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs,

etc.), especially humans, the dosage varies depending on the

symptoms, age, gender, weight, sensitivity difference of the

patient, administration method, administration interval,

type of active ingredient, and type of formulation, and is

not particularly limited, but for example, 30 pg-100 g, 100

pg-500 mg, or 100 pg-100 mg may be administered once or

several times. In case of the injection administration,

depending on the weight of patient, 1 pg/kg-3000 pg/kg, 3

pg/kg-1000 pg/kg may be administered once or several times.

[0093] The methods of preventing or treating influenza using the peptides of the present invention may be carried out with reference to the description for the above medical drug compositions.

[0094] Another embodiment disclosed in this description

relates to a virus detection agent, in particularly an

influenza virus detection agent. This virus detection agent

contains the above-mentioned hemagglutinin-binding peptide,

the salt thereof, or the solvate thereof.

[0095] (Virus detection agents and detection kits)

The present invention also encompasses virus detection

agents, in particularly influenza virus detection agents,

containing the peptides of the present invention. The

peptides of the present invention specifically bind to a

hemagglutinin on the surface of a virus. Therefore, for

example, the peptides of the present invention may be used

in place of anti-influenza antibodies in immunoassays such

as ELISA method to detect influenza viruses in a sample.

The peptides of the present invention may be detectably

labeled if used as a detection agent. The peptides may be

labeled with known labeling substances, for example, the

peptides labeled with enzymes such as peroxidase and alkaline

phosphatase, radioactive substances such as 1251, 1311, 35S

and 3H, fluorescent substances such as fluorescein

isothiocyanate, rhodamine, dansyl chloride, phycoerythrin,

tetramethyl rhodamine isothiocyanate, and near-infrared fluorescent substances, and luminescent substances such as luciferase, luciferin, and equilin are used. In addition, the peptides labeled with nanoparticles such as gold colloids, quantum dots may also be detected.

In addition, in immunoassays, the peptides of the

present invention may be labeled with biotin and bound to

avidin or streptavidin labeled with enzymes, etc. for

detection.

Among immunoassays, ELISA method using enzymatic

labeling is preferable because antigens may be measured

easily and quickly. For example, an antibody that

specifically recognize a moiety of an influenza virus other

than the hemagglutinin are fixed to a solid phase carrier,

and a sample is added and reacted, followed by the labeled

peptides of the present invention is added and reacted. The

influenza virus may be detected by washing it and then

reacting it with an enzyme substrate, developing a color,

and measuring the absorbance. After reacting the antibody

immobilized on the solid phase carrier and the sample,

unlabeled peptides of the present invention are added, and

an antibody to the peptides of the present invention may be

enzymatically labeled and further added. In addition, the

peptides of the present invention are immobilized to a solid

phase carrier as a capture substance, and the labeled

antibodies that recognize influenza viruses may be used as

a detection substance, or the peptides of the present

invention may be used for either capture or detection.

In the enzyme substrate, if the enzyme is peroxidase,

3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine

(TMB), o-phenylenediamine (OPD), etc. may be used, and if

the enzyme is alkaline phosphatase, p-nitrophenyl phosphate

(NPP), etc. may be used.

[0096] Herein, the "solid phase carrier" is not particularly

limited as long as it is a carrier on which the peptides may

be immobilized, and includes microtiter plates made of glass,

metal, resin, etc., substrates, beads, nitrocellulose

membranes, nylon membranes, PVDF membranes, and the target

substances may be immobilized on these solid phase carriers

according to known methods.

[0097] The inspection kit according to the present invention

includes reagents and instruments necessary for the above

detection (including, but not limited to, peptides of the

present invention, antibodies, solid phase carriers, buffer

solutions, enzyme reaction termination solutions, microplate

readers).

[0098] Another embodiment disclosed in this description may

also be considered for use as an influenza virus detection

kit containing the above-mentioned influenza virus detection

agent, or as a tool to elucidate hemagglutinin-mediated

influenza virus infection, and various cellular functions

and life phenomena associated with it.

[00991 This description also provides the use of

hemagglutinin-binding peptide to produce a medical drug for

the prevention or therapy of influenza. In this case, the

hemagglutinin-binding peptide may be any of the above

mentioned ones.

[0100] The present description also provides a method of

preventing or treating influenza including the step of

administering an effective amount of the hemagglutinin

binding peptide, the pharmaceutically acceptable salt

thereof, or the solvate thereof as an active ingredient to

a subject that is a human, a non-human mammal, or an avian.

As the hemagglutinin-binding peptide, the above-mentioned

peptides may be used as appropriate. Examples of the non

human mammal are non-human primates, pigs, cattle, dogs,

cats, horses, sheep, rats and mice.

[0101] The abbreviations used herein, particularly in the

following representative examples, are well known to those

skilled in the art. Some of the abbreviations used are as

follows: Fmoc as 9-fluorenylmethyloxycarbonyl; HOAt as 1

hydroxybenzotriazole; HATU as 0-(7-azabenzotriazol-1-yl)

N,N,N',N'-tetramethyluronium hexafluorophosphate; MeCN as

acetonitrile; DBU as 1,8-diazabicyclo "5.4.0"-7-undecene;

DIPEA as N,N-diisopropylethylamine; DODT as 3,6-dioxa-1,8

octane-dithiol; DMSO as dimethyl sulfoxide; DMF as N,N-

Dimethylformamide; mL as milliliter (unit); M as molar

(unit); Mtt as monomethyl trityl; Mmt as monomethoxy trityl;

o-Ns as 2-nitrobenzenesulfonyl; v/v as volume/volume; TFA as

trifluoroacetic acid; TIS as triisopropylsilane; Trt as

trityl.

[0102] Examples and general methods

All the raw materials, building blocks, reagents,

acids, bases, solid phase resins, and solvents used in the

chemical synthesis of the present invention were either

commercially available or may be synthesized by those skilled

in the art using organic chemical methods. Unless otherwise

specified, amino acids containing protecting groups were

used as they are commercially available products.

[0103] In the present invention, a structure determination

of the chemically synthesized peptides was confirmed by ESI

MS(+) in the mass spectrum analysis method with molecular

weights calculated considering amino acids used according to

the target sequence and building blocks used as needed. The

term "ESI-MS(+)" means an electrospray ionization mass

spectrum analysis method carried out in the positive ion

mode. The detected masses were reported in "m/z" units.

Compounds with molecular weights greater than approximately

1000 were detected frequently as divalent or trivalent ions.

[0104] In the present invention, the purity of the chemically synthesized peptides was determined by one of the following analytical methods.

(Analysis conditions)

Analysis condition A

Column: CORTECS (registered trademark) UPLC (registered

trademark) C18 column (Nihon Waters), 90 A, 1.6 pm, 2.1 x

100 mm

Mobile phase: MeCN/0.025% TFA in H 2

Temperature: 400C

Gradient: 5-95% MeCN/0.025% TFA in H2 in 5.56 min; linear

gradient

Flow rate: 0.4 mL/min

Detection method: UV 220 nm

Analysis condition B

Column: Kinetex EVO C18 2.6 pm, 2.1 ID x 150 mm, 100

A(Phenomenex)

Column temperature: 600C

Mobile phase A: 0.025% TFA in H 2

Mobile phase B: 0.025% TFA in CH 3 CN

Gradient: as described in each example

Flow velocity: 0.25 mL/min

Detection: PDA (225 nm)

[0105] Elongation of the peptide chain in the solid phase

resin described in the present invention was carried out using the resin described in each example as a starting material and using peptide coupling reaction conditions and

Fmoc removal reaction conditions normally used. The

reaction was carried out using CEM Liberty Blue, an automated

peptide synthesizer, according to the manufacturer's manual.

Common amino acids used are listed below, and side chain

protecting groups are shown in parentheses.

Fmoc-Trp(Boc)-OH; Fmoc-Thr(tBu)-OH; Fmoc-N-Me-Gly-OH; Fmoc

Asp(OtBu)-OH; Fmoc-N-Me-Phe-OH; Fmoc-Ala-OH; Fmoc-N-Me-Ala

OH; Fmoc-His(Trt)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH; Fmoc

HydPro(tBu)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Lys(Mtt)-OH; Fmoc

Ser(tBu)-OH; Fmoc-N-Me-Ser(tBu)-OH.

[0106] The introduction of the chloroacetyl group was carried

out by removing the Fmoc group of the a-amino group from the

solid phase resin holding the Fmoc-protected peptide

obtained in the previous step by the method described above,

followed by adding chloroacetic acid (about 3 equiv.), about

3 equiv. of N,N'-diisopropylcarbodiimide in DMF solution

(0.5 M), and about 3 equiv. of HOAt in DMF solution (0.5 M)

and shaking at room temperature for 40 minutes.

For deprotection of the side chain and cutting out from

the solid phase resin, the resin obtained after the

chloroacetyl group introduction step was first washed five

times with DMF and methylene chloride, respectively, and

dried under reduced pressure. Subsequently, reactant

cocktail- A (a mixture of TFA/H 2 /TIS/DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid phase resin and shaken at room temperature for 150 minutes. The reaction solution was collected from the frit by filtration. The solid phase resin remaining in the reaction vessel was shaken again with the cut-out cocktail, and the solution component was collected from the frit and mixed with the above-mentioned filtrate.

If this filtrate was added to an excess of diethyl ether

cooled to 0°C, a cloudy precipitate formed. This mixture

was centrifuged (9000 rpm, 3 min) and the solution was

decantated. The resulting solid was washed again with a

small amount of diethyl ether cooled to 0°C, and then the

resulting solid was used for the next cyclization reaction.

[0107] In the present invention, the cyclization reaction of

the peptides was carried out by dissolving the peptides in

DMSO to a final concentration of 5 mM, based on the number

of moles of the solid phase resin, and then adding 6 equiv.

of triethylamine and stirring at room temperature for about

16 hours. The resulting reaction solution was acidified

with acetic acid and concentrated under reduced pressure

using Biotage (registered trademark) V-10 (Biotage Japan).

As the method for purifying the resulting crude

purified peptides, reversed-phase preparative HPLC on Waters

Auto Purification System - SQD2 single quadruple mass

spectrometer was used, elution was carried out while

monitoring m/z ion derived from the target object. It was confirmed that the mass spectrum obtained in the scan mode of ESI-positive and the mass spectrum containing the multivalent ions calculated from the molecular formula of the target object match within the error range of the mass spectrometer used. The purification conditions, including the columns used, are shown in each example.

<Example 1>

[0108] Synthesis of HA119

[Formula 32]

OHO0 oH H 0 N N N 0

0 NH 0 N HO 0o N O0 0 NH H

H2 N HN ' N

0 HN O HN 0_ HO1 00 N~ NH HNN N N 0 H Hd HO

[0109] Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.15 mmol/g,

0.43 g) was used to synthesize the target peptide, starting

with the removal of Fmoc according to the above-mentioned

general method. The chloroacetyl group was subsequently

introduced according to the general method.

[0110] The resulting crude product was purified using the

following conditions (column: Waters Xbridge (registered

trademark) C18 5 pm OBD (registered trademark) 19 x 150 mm

(Nihon Waters); mobile phase: A = 0.1% TFA in H 2 0, B = 0.1%

TFA in MeCN; temperature: 400C; gradient (%B): 5-29% over 3

min, then 29-34% over 8 min; flow rate: 17 mL/min).

[0111] The purity of the target object was calculated from

the area ratio of the LC/MS (UV wavelength 225 nm)

chromatogram under the analytical condition B, and was 99.4%.

Analysis condition A: retention time=3.57 min, ESI-MS(+)

observed value m/z= 899.8, theoretical value 899.5 ((M/2)+H)

Analysis condition B: retention time = 16.5 min; gradient

(%B conc): 25-65% over 20 min, then 65-95% over 1 min, then

95% over 5 min.

<Example 2>

[0112] Synthesis of HA146

[Formula 33]

HH K N N N O

0 NH 0 N' N o HO N 0 NH S, H

H H N 4 N NHN N 0 H2 N 0 0 HN 0 HN 0 HO,,

N N NHHN'

H H Hd

' HO

[0113] Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.37 mmol/g,

1.35 g) was placed in a reaction vessel with a frit, shaken

with dichloromethane and allowed to expand. Fmoc-Lys(Mtt)

OH was introduced into the resulting solid phase resin by

peptide coupling after removal of Fmoc according to the

above-mentioned general method. The resulting solid phase

resin was swollen with dichloromethane, reactant cocktail-B

(TFA/TIS/CH 2 Cl 2 by volume ratio 1:4:95) was added, the solid

phase resin was shaken at room temperature for 30 min, and

then the reaction solution was drained from the frit. After

repeating this operation 12 times, the color of the filtrate

became colorless and transparent. This point was considered

the completion of the reaction. To the resulting solid phase

resin, a DMF solution of decanoic acid (0.21 M, 12 mL), a

DMF solution of HATU (0.5 M, 5 mL), and a DMF solution of

DIPEA (1 M, 5 mL) were added, and the mixture was shaken at

°C for 40 min. After the reaction solution was drained

from the frit, the resulting solid phase resin was washed

with DMF, followed by dichloromethane.

[0114] Fmoc was removed from the solid phase resin obtained

by the above operation, and Fmoc-amino acids were

sequentially introduced utilizing the automated synthesizer

in the above-mentioned general method. The amino acid and

reagent used in the reaction were calculated in equal amounts,

assuming that the solid phase resin was 0.5 mmol. The

peptide coupling was carried out by the automated synthesizer,

subsequently, chloroacetyl group was introduced according to

the above-mentioned general approach.

Subsequently, the resulting solid phase resin was used

to deprotect the side chain, cut out from the solid phase

resin, and carry out a cyclization reaction according to the

above-mentioned general method.

The resulting crude product was purified using the

following conditions (column; Waters Xbridge (registered

trademark) C18 5 pm OBD (registered trademark) 50 x 250 mm

(Nihon Waters); mobile phase: A = 0.1% TFA in H 2 0, B = 0.1%

TFA in MeCN; temperature: 400C; gradient (%B): 16-41% over

3 min, then 47-52% over 7 min, then 47-80% over 1.5 min;

flow rate: 120 mL/min).

[0115] The purity of the target object was calculated from the area ratio of the LC/MS (UV wavelength 225 nm) chromatogram under the analytical condition B, and was 99.3%.

Analysis condition A: retention time = 4.50 min, ESI-MS(+)

observed value m/z = 1040.4, theoretical value 1040.2

((M/2)+H)

Analysis condition B: retention time = 18.8 min; gradient

(%B conc): 25-65% over 20 min, then 65-95% over 1 min, then

95% over 5 min.

<Example 3>

[0116] Synthesis of HA145

[0117] [Formula 34]

OHO H N N 0 N 0 NH 0 N HO N"I- 0 0 NH

H Ho N N NI HIN N H ' N H 0 H2N 0 O HN O HN 0 HO 0 0 H H" 0HN'

N N N HH HO 1 HO

HA145 was synthesized according to the synthesis

method shown in Example 1, using lauric acid instead of

decanoic acid.

The resulting crude product was purified using the

following conditions (column: Waters Xbridge (registered trademark) C18 5 m OBD (registered trademark) 50 x 250 mm

(Nihon Waters); mobile phase: A = 0.1% TFA in H 2 0, B = 0.1%

TFA in MeCN; temperature: 400C; gradient (%B): 21-46% over

3 min, then 46-51% over 7 min, then 51-80% over 1.5 min;

flow rate: 120 mL/min).

The purity of the target object was calculated from

the area ratio of the LC/MS (UV wavelength 225 nm)

chromatogram under the analytical condition B, and was 99.3%.

Analysis condition A: retention time = 4.42 min, ESI-MS(+)

observed value m/z = 1055.1, theoretical value 1054.3

((M/2)+H)

Analysis condition B: retention time = 18.8 min, gradient

(%B conc): 25-65% over 20 min, then 65-95% over 1 min, then

95% over 5 min.

<Example 4>

[0118] Synthesis of HA152

[Formula 35]

OH N HN N 0 NH 0 NHN o1j H 0 HO 0 N H 0 aH 0 H SIH'1 N NHHN ". N N H 11i 00 1)" HNc0 HN 0 0HO"( 00 H H_ NH HN

[0119]~~ ~ PAL-PE resi (Wtnb Chmcl020ml/,11 HON

HO

[0119] PAL-PEG resin (Watanabe Chemical, 0.22 mmol/g, 1.16

g) was placed in a reaction vessel with a frit, shaken with

dichloromethane and allowed to expand. After draining from

the dichloromethane frit, a dichloromethane solution (5 mL)

of 2-nitrobenzenesulfonyl chloride (4 equiv.) and a

dichloromethane solution (4 mL) of DIPEA (4 equiv.) were

added and the mixture was stirred at room temperature for 30

min. After the reaction solution was drained from the frit,

the solid phase resin was washed with dichloromethane. To

the resulting solid phase resin, a THF solution (6 mL) of

Fmoc-glycinol (10 equiv.), a dichloromethane solution (5 mL)

of triphenylphosphine (12 equiv.), and a dichloromethane

solution (5 mL) of diisopropyl azodicarboxylate (10 equiv.)

were added and the mixture was shaken at room temperature.

The progress of the reaction was confirmed by LCMS after a

small amount of the solid phase resin was taken out and

treated with a cut-out cocktail. After the reaction solution was drained from the frit, the solid phase resin was washed with dichloromethane. To the resulting solid phase resin, a DMF solution (12 mL) of piperidine (20%) was added and the mixture was shaken at room temperature for 30 min. The reaction solution was drained from the slit, and the DMF solution (12 mL) of piperidine (20%) was added again and the mixture was shaken at room temperature for 30 min. After the reaction solution was drained from the slit, the resulting solid phase resin was washed with DMF, followed by dichloromethane. To the resulting solid phase resin, a DMF solution of decanoic acid (0.21 M, 5 mL), a DMF solution of

HATU (0.5 M, 2 mL), and a DMF solution of DIPEA (1 M, 2 mL)

were added, and the mixture was shaken at 400C for 1 hour.

After the reaction solution was drained from the frit, the

resulting solid phase resin was washed with DMF, followed by

dichloromethane. The resulting solid phase resin was

immersed in 10 mL of DMF, and DODT (0.4 mL, 10 equiv.) and

DBU (0.37 mL, 10 equiv.) were added, and the mixture was

stirred at room temperature. The progress of the reaction

and the disappearance of the raw material were confirmed by

LCMS after treating a small amount of the solid phase resin.

[0120] Fmoc-amino acids were sequentially introduced into

the solid phase resin obtained by the above operation,

utilizing the automated synthesizer in the above-mentioned

general method. The amino acid and reagent used in the

reaction were calculated in equal amounts, assuming that the solid phase resin was 0.25 mmol. The peptide coupling was carried out by the automated synthesizer, subsequently, chloroacetyl group was introduced according to the above mentioned general approach.

[0121] The resulting solid phase resin was used to deprotect

the side chain, cut out from the solid phase resin, and carry

out a cyclization reaction according to the above-mentioned

general method.

[0122] The resulting crude product was purified using the

following conditions (column: Waters Xbridge (registered

trademark) C18 5 pm OBD (registered trademark) 50 x 250 mm

(Nihon Waters); mobile phase: A = 0.1% TFA in H 2 0, B = 0.1%

TFA in MeCN; temperature: 400C; gradient (%B): 17-42% over

3 min, then 42-47% over 7 min, then 47-80% over 1.5 min;

flow rate: 120 mL/min).

The purity of the target object was calculated from

the area ratio of the LC/MS (UV wavelength 225 nm)

chromatogram under the analytical condition B, and was 98.4%.

[0123] Analysis condition A: retention time = 4.18 min, ESI

MS(+) observed value m/z = 998.6, theoretical value 998.2

((M/2)+H).

Analysis condition B: retention time = 16.48 min, gradient

(%B conc): 25-65% over 20 min, then 65-95% over 1 min, then

95% over 5 min.

<Example 5>

[0124] Synthesis of HA151

[Formula 36]

OH 0 O

NN NNN H H 0 N 0 NH HOH-T N" 0 0 NH rHH 0 H 7 ".~

H HN G N N HI N0 N "r H H HNH HO

' N N HO, 0 HH

[0125]N HA15 wa sythsie accodn totesNthsi

H6 HO

[0125] HA151 was synthesized according to the synthesis

method shown in Example 4, using lauric acid instead of

decanoic acid.

[0126] The resulting crude product was purified using the

following conditions (column; Waters Xbridge (registered

trademark) C18 5 pm OBD (registered trademark) 50 x 250 mm;

mobile phase: A = 0.1% TFA in H 2 0, B = 0.1% TFA in MeCN;

temperature: 400C; gradient (%B): 22-47% over 3 min, then

47-52% over 7 min, then 52-80% over 1.5 min; flow rate: 120

mL/min).

[0127] The purity of the target object was calculated from the area ratio of the LC/MS (UV wavelength 225 nm) chromatogram under the analytical condition B, and was 97.7%.

Analysis condition A: retention time = 4.49 min, ESI-MS(+)

observed value m/z = 1012.4, theoretical value 1012.2

((M/2)+H)

Analysis condition B: retention time = 12.5 min, gradient

(%B conc): 40-80% over 20 min, then 80-95% over 1 min, then

95% over 5 min.

<Example 6>

[0128] [Evaluation of the anti-virus activity of the peptides

against influenza viruses.]

In order to confirm the in vitro anti-virus activity

of the peptides against influenza viruses, the test was

carried out by the method shown below. The specific test

method is shown below.

1) MDCK cells were seeded at 3x104 cells/well and

cultured at 370C, 5% C02, in the presence of MEM-10% FBS for

24 hours.

2) after incubation, 100 pL of serum-free MEM was added

to the well and the cell monolayer was washed.

3) the compounds to be tested were dissolved by an

infection maintenance medium (serum-free MEM containing

vitamins) and adjusted to each measured concentration.

4) a test compound dissolved in the serum-free MEM that

is the infection maintenance medium were added to each well.

5) Influenza viruses A/Nagasaki/HA-58/2009 (H1N1),

A/Puerto Rico/8/34 (H1N1), or A/Duck/Pennsylvania/84 (H5N2)

was diluted with the infection maintenance medium containing

trypsin to prepare 1,000 TCIDso/mL.

6) the diluted virus solution was added at 100 pL/well

and the titer per well was adjusted to 100 TCID5 o.

7) the viruses were incubated at 370C, 5% C02

conditions for 72 hours.

8) after completion of incubation, the culture medium

was removed from each well.

9) a 70% ethanol aqueous solution was added at 200

ul/well and allowed to stand still at room temperature for

5 minutes.

10) after removing the ethanol aqueous solution, a 0.5%

crystal violet aqueous solution was added at 200 pL/well and

allowed to stand still at room temperature for 5 minutes.

11) it was rinsed with water and dried under room

temperature.

12) TECAN infinite 200 (TECAN) was used to measure the

absorbance of each well at the measurement wavelength of X

= 560 nm.

13) at each concentration, the relative value (CV

relative value, %) was calculated when the mock group (drug

free and virus-free group) was set at 100%.

14) GraphPad Prism 5.0 (GraphPad Software) was used to

determine the EC50 value of each specimen.

[0129] In order to confirm the in vivo anti-virus activity of the peptides against influenza viruses, the test was carried out by the method shown below. The specific test method is shown below.

1) five BALB/cA Jcl[SPF] mice per group were adjusted

as infection model mice.

2) the virus solution stored at -80°C was gently melted

on ice, centrifuged for a few seconds, and then dispensed

into a tube containing PBS.

3) the anesthetized mice were nasally inoculated with

influenza virus A/Puerto Rico/8/34 (H1N1) at 267 pfu per

mouse, and the above-mentioned inoculation time point was

set as "day 0".

4) HA152 was dissolved in a 10% hydroxypropyl-B

cyclodextrin solution that is a dissolving solvent.

5) Peramivir was dissolved in the PBS solution.

6) Peramivir and HA152 were administered intravenously

in the tail at a dose of 30 pmol/kg and 15 pmol/kg,

respectively. As a vehicle control, the 10% hydroxypropyl

B-cyclodextrin solution was administered intravenously in

the tail as well.

7) after influenza virus infection and compound

administration, the condition was observed once a day,

including life or death.

8) the survival rate was calculated by converting the

survival rate for 14 days with the day of infection as day

0.

[0130] 3. Result

Results of HA152 activity evaluation using influenza viruses

A/Nagasaki/HA-58/2009 (H1N1), A/Puerto Rico/8/34 (H1N1), and

A/Duck/Pennsylvania/84 (H5N2)

The results of in vitro evaluation of anti-virus

activities of iHA100 and HA152 against influenza viruses

A/Nagasaki/HA-58/2009 (H1N1), A/Puerto Rico/8/34 (H1N1), and

A/Duck/ Pennsylvania/84 (H5N2) are shown in Figure 1. iHA100

inhibited virus-induced cell death in the high concentration

range of 1 pM or more in the final concentration, but did

not show remarkable inhibition of cell death in the low

concentration range of nM range. On the other hand, HA152

was found to show remarkable inhibition of cell death even

in low concentration range of pM or less, and have remarkable

anti-virus activity against both human and avian test

influenza viruses, even in low concentration range. This

confirmed that HA152 showed extremely high anti-influenza

virus activity compared to iHA100.

[0131] Next, using influenza virus A/Duck/Pennsylvania/84

(H5N2), the in vitro anti-virus activity of the compounds

containing iHA100 and HA152 was calculated as EC50. As shown

in Figure 2, the compound containing HA152 showed at least

10 times higher anti-virus activity than iHA100. Therefore,

it was shown that HA152 and each compound shown in Fig. 2

retains remarkable higher anti-virus activity compared to

iHA100.

[0132] Results of HA152 activity evaluation using the

influenza virus A/Puerto Rico/8/34 (H1N1) in a mouse

infection model

The results of in vivo anti-virus activity evaluation

of peramivir and HA152 using the influenza virus A/Puerto

Rico/8/34 (H1N1) are shown in Figure 3.

An analysis of the survival rate of mice infected with

the influenza virus over 14 days showed that the survival

rate for the group that did not receive the drug was 0%. On

the other hand, the survival rate of the group receiving a

single dose of 30 pmol/kg of peramivir was 20%, whereas the

survival rate of the group receiving 15 pmol/kg of HA152 was

60%. This indicates that HA152 exhibited remarkable anti

virus activity not only in vitro, but also in vivo. Its

anti-virus activity was comparable or superior to peramivir

that is an approved pharmaceutical product that targets

neuraminidase.

INDUSTRIAL APPLICABILITY

[0133] This invention may be utilized in the medical drug

and medical device industries.

SEQUENCE LISTING

<110> PeptiDream INC. <120> Hemagglutinin‐Binding Proteins

<130> 18‐134P

<160> 7

<170> PatentIn version 3.5

<210> 1 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<400> 1

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15

<210> 2 <211> 16

<212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<400> 2

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15

<210> 3 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (1)..(1) <223> N‐chloroacetyl‐L‐tryptophan

<220> <221> MOD_RES

<222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<220> <221> MOD_RES <222> (15)..(15) <223> AMIDATION

<400> 3

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15

<210> 4 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (1)..(1) <223> N‐chloroacetyl‐L‐tryptophan

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220>

<221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<220> <221> MOD_RES <222> (16)..(16) <223> Lys[gamma‐C(=O)n‐C11H23]‐NH2

<400> 4

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15

<210> 5 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (1)..(1) <223> N‐chloroacetyl‐L‐tryptophan

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<220> <221> MOD_RES <222> (16)..(16) <223> Lys[gamma‐C(=O)n‐C9H19]‐NH2

<400> 5

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15

<210> 6 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (1)..(1) <223> N‐chloroacetyl‐L‐tryptophan

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8) <223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<220> <221> MOD_RES <222> (15)..(15) <223> Cys‐[NHCH2CH2NHC(=O)n‐C11H23]

<400> 6

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15

<210> 7 <211> 15 <212> PRT <213> Artificial Sequence

<220> <223> Synthesized polypeptide

<220> <221> MOD_RES <222> (1)..(1) <223> N‐chloroacetyl‐L‐tryptophan

<220> <221> MOD_RES <222> (3)..(3) <223> N‐methylglycine

<220> <221> MOD_RES <222> (5)..(5) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (6)..(6) <223> N‐methylphenylalanine

<220> <221> MOD_RES <222> (8)..(8)

<223> N‐methylalanine

<220> <221> MOD_RES <222> (13)..(13) <223> 4‐Hydroxy‐L‐proline

<220> <221> MOD_RES <222> (15)..(15) <223> Cys‐[NHCH2CH2NHC(=O)n‐C9H19]

<400> 7

Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15

Claims (12)

1. A hemagglutinin-binding peptide, a pharmaceutically

acceptable salt thereof, or a solvate thereof, wherein the

hemagglutinin-binding peptide is:

(1) a polypeptide consisting of an amino acid sequence

represented by sequence no. 1 or 2:

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val

hydPro-Ala-Cys (sequence no. 1), and

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val

hydPro-Ala-Cys-Lys (sequence no. 2);

(2) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1 or 2, the N-terminal Trp is

chloroacetyl-Trp;

(3) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the N-terminal Trp is

chloroacetyl-Trp and the C-terminal Cys has been modified as

represented by Formula (I) via an amide bond;

(4) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the N-terminal Trp is chloroacetyl

Trp and the C-terminal Lys has been substituted with a

modified lysine derivative represented by Formula (II);

(5) a polypeptide consisting of an amino acid sequence

in which, in the sequence no. 1, the C-terminal Cys has been

modified as represented by Formula (I) via an amide bond;

(6) a polypeptide consisting of a sequence comprising

a lysine derivative represented by Formula (II) in which, in the sequence no. 2, a side chain of Lys has been modified with an acyl group; or

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1) to (6) above, one

or two amino acids have been deleted, added, substituted, or

inserted (however a sequence in which, in the sequence no.

1, the C-terminal Cys has been deleted, and a sequence in

which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded).

Formula (I)

[Formula 1]

H A1 NN H 0

(In Formula (I), * denotes a linking moiety to a carbonyl

group of the C-terminal Cys, and A' denotes a C8-C12 alkyl

group.)

Formula (II)

[Formula 2]

0 NH 2

N NH

H A1 0

(In Formula (II), * denotes a linking moiety to a carbonyl

group of the C-terminal Cys, and A' denotes a Cs-C12 alkyl

group.)

2. The hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to claim 1, wherein the hemagglutinin

binding peptide is:

(1) the polypeptide consisting of the amino acid

sequence represented by the sequence no. 1 or 2:

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val

hydPro-Ala-Cys (sequence no. 1), and

Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val

hydPro-Ala-Cys-Lys (sequence no. 2);

(5) the polypeptide consisting of the amino acid

sequence in which, in the sequence no. 1, the C-terminal Cys

has been modified as represented by Formula (I) via an amide

bond;

(6) a polypeptide consisting of a sequence in which,

in the sequence no. 2, the C-terminal Lys has been substituted with a modified lysine derivative represented by

Formula (II); or

(7) a peptide having an amino acid sequence in which,

in the amino acid sequence in any of (1), (5) and (6) above,

one or two amino acids have been deleted, added, substituted,

or inserted (however the sequence in which, in the sequence

no. 1, the C-terminal Cys has been deleted, and the sequence

in which, in the sequence no. 2, the C-terminal Lys has been

deleted, are excluded).

3. The hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to claim 1, wherein the hemagglutinin

binding peptide is cyclic.

4. The hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to claim 3, wherein the hemagglutinin

binding peptide is represented by the following Formula (III)

or (IV).

[Formula 3]

OH

OH

NH N N HN N SHH 0 0 0 OH 0 NH

0 NH 0

HN 00 OHN0

N 0SN

N N HN0 H H o

0 0 OH

(In Formula (III), Formula A 2 denotes a group represented by

-NH 2 or a group represented by Formula (I).)

[Formula 4]

0 OH A3 S -"y 0 HNH HN 0

HN

HN:: / HO 0 1,NH NH OHN 0

HOCf O 0 N N 0 N (IV)

NH OHN 0

0 0

HO HN 0O NH 0 H IN N N 0 H H 0 N

N

(In Formula (IV), Formula A 3 denotes a group represented by

-NH 2 or Formula (II). )

5. The hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to claim 3, wherein the hemagglutinin

binding peptide is represented by the following Formula (V)

or (VI).

[Formula 5]

OH

OH

N- 0

NH N N HN N H H 00 OAO *0 NH

0 NH 0 O

NN HN A4

N~r NH

HN 0 ,, N

N OH 0 N FIN : 0

N N 0 N H i 0 0 OH

(In Formula (V), Formula A 4 denotes a group represented by

NH 2 or a group represented by Formula (IIa).)

[Formula 6]

o NH 2

N NH H

A1 O(Ia)

(In Formula (IIa), * denotes a linking moiety, and A' denotes

a C8-C 1 2 alkyl group.)

[Formula 7]

0 OH 0 S 0

HN 0 A5 -N NH O

00

N 0 N (VI)

NH OH N 0

0 0 HO 0 NH 0 HI

N O

N

In Formula (VI), Formula A 5 denotes a group represented by

NH 2 or Formula (I) .

6. The hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to claim 3, wherein the hemagglutinin

binding peptide is represented by the following Formula (VII)

[Formula 8]

0

HN

NH S O O0 O

HN N O NH H N HN HO HN O'NH

HO"' 0 0O" N 0 <.N

NH OH N 0 H:7 0

HO HN NH

<N O

NN

7. A medical drug for prevention of virus infection or

for therapy of a virus infectious disease comprising the

hemagglutinin-binding peptide, the pharmaceutically

acceptable salt thereof, or the solvate thereof according to

any of claims 1 to 6.

8. A medical drug for prevention or therapy of influenza

comprising the hemagglutinin-binding peptide, the

pharmaceutically acceptable salt thereof, or the solvate

thereof according to any of claims 1 to 6.

9. A virus detection agent comprising the hemagglutinin- binding peptide, the pharmaceutically acceptable salt thereof, or the solvate thereof according to any of claims

1 to 6.

10. An influenza virus detection agent comprising the

hemagglutinin-binding peptide, the pharmaceutically

acceptable salt thereof, or the solvate thereof according to

any of claims 1 to 6.

11. A kit for virus detection comprising the virus

detection agent according to claim 9.

12. A kit for influenza virus detection comprising the

influenza virus detection agent according to claim 10.